Palmitoylation refers to a dynamic post-translational modification of proteins involving the covalent attachment of long-chain fatty acids to the side chains of cysteine, threonine or serine residues. In recent years, palmitoylation has been identified as a widespread modification of both viral and cellular proteins. Because of its dynamic nature, protein palmitoylation, like phosphorylation, appears to have a crucial role in the functioning of the nervous system. Several important questions regarding the post-translational acylation of cysteine residues in proteins are briefly discussed: (a) What are the molecular mechanisms involved in dynamic acylation? (b) What are the determinants of the fatty acid specificity and the structural requirements of the acceptor proteins? (c) What are the physiological signals regulating this type of protein modification, and (d) What is the biological role(s) of this reaction with respect to the functioning of specific nervous system proteins? We also present the current experimental obstacles that have to be overcome to fully understand the biology of this dynamic modification.

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20-and 60-day-old rats were incubated with [ 3 H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 M), or rotenone (10 M) reduced the incorporation of [ 3 H]palmitate into fatty acylCoA and glycerolipids by 50 -60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 M) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [ 3 H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains covalently bound fatty acids, predominantly palmitic acid. This study adapts a stable isotope technique (Kuwae, T., Schmid, P. C., Johnson, S. B., and Schmid, H. O. (1990) J. Biol. Chem. 265, 5002-5007) to quantitatively determine the minimal proportion of PLP molecules which undergo palmitoylation. In these experiments, brain white matter slices from 20-day-old rats were incubated for up to 6 h in a physiological buffer containing 50% H 2 18 O. The uptake of 18 O into the carbonyl groups of fatty acids derived from PLP, phospholipids, and the free fatty acid pool was measured by gas-liquid chromatography/mass spectrometry of the respective methyl esters. Palmitic acid derived from PLP acquired increasing amounts of 18 O, ending with 2.9%18 O enrichment after 6 h of incubation.18 O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18 O enrichment). After correcting for the specific activity of the 18 O-enriched free palmitic acid pool, 7.6% of the PLP molecules were found to acquire palmitic acid in 6 h. This value is not only too large to be the result of the palmitoylation of newly synthesized PLP molecules, it was also unchanged upon the inhibition of protein synthesis with cycloheximide.18 O enrichment in less actively myelinating 60-day-old rats was significantly reduced. In conclusion, our experiments suggest that a substantial proportion of PLP molecules acquire palmitic acid via an acylation/deacylation cycle and that this profile changes during development.A number of integral membrane proteins are modified after their synthesis by the covalent attachment of long-chain fatty acids (mostly palmitic acid) to one or more cytoplasmically oriented cysteine residues (for review, see Refs. 1-6). In the majority of the cases, the chemically bound acyl chains turn over much faster than the protein backbone, implying that palmitoylation is a regulatory modification. In fact, fatty acylation of this and other types of proteins has been shown to be modulated by physiological (7-9) or pharmacological stimuli (10 -14). To date, the metabolic features of palmitoylation have only been studied by labeling cultured cells with [ 3 H]palmitic acid, and the half-life of the palmitate has been estimated from the disappearance of the protein-bound radioactivity after isotopic dilution with the unlabeled fatty acid. Unfortunately, labeling experiments using [ 3 H]palmitic acid are difficult to interpret, particularly when considering the possibility that exogenous and endogenous palmitate may not have equal access to the fatty acid donor pools. Furthermore, since the specific radioactivity of the donor pool of palmitate used for protein palmitoylation cannot be estimated, it is not possible to determine the number of protein molecules participating in such rapid deacylation-reacylation cycles. Consequently, the radioactivity that becomes associated with a polypeptide during the course of an...

In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60‐day‐old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 µM) reduced the incorporation of [3H]palmitate into all brain glycerolipids by 40–50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [3H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 µM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage‐gated sodium channels and involves the stimulation of the Na+/K+ pump. Decreased levels of both ATP, due to activation of the Na+,K+‐ATPase, and the precursor palmitoyl‐CoA were found in the veratridine‐treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage‐dependent Na+ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K+]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.

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